Immunoscreening And Expression The High Reactionogenicity Antigen Of Clonorchiasis

Clonorchiasis also called as liver flukesd isease, caused by Clonorchis sinensis, is a zoonosis widely distributes in East Asia and most parts of China. It is estimated that about 15 million people are infected in China. The adults of C.sinensis Parasitize in the liver-biliary ductssy system of human or other hosts and then do chronic impairment Such as bile duct regional distension,bile duct epithelial porliferation,cholangitis,cirrhosis, even induce bile duct epithelial cells cancer and liver cancer. Clonorchiasis is listed as one of significant infectious disease of our country,attracting great attentions aroused the country.That accurate diagnosis of clonorchiasis is an important problem that is confornted in prevention and clinic fields. At present, the diagnostic methods of C.sinensis infection are depended on pathogen examination and immunological detcetion.The tradditional light microscopy for eggs in feces easily missed diagnosis and misdiagnosises and is time-labour wasting, The immunological diagnostic methods for Clonorchiasis with high false negative rate and false positive rate.The source of crude antigen is limited and difficult to standardize.So, Finding and screening of diagnostic recombinant antigen is the major task of C.sinensis research. ELISA were used for the quantitative determination of antibodies against the adult antigen of C.sinensis in sera 1w, 10d, 2w, 3w, 4w,5w ,7w, 9w, 11w, 13w, 15w after infection. The antibodies in rabbits and mice increased at the 2th - 9th week after infection, which reached the peak at the 10th week and keep up to 15week.Immunological screening of the cDNA library was performed. 23 positive clones were get, which seven clones rich in glycine 87% homologous with the one submission by scientist of Korea,six clones rich in proline 55-84% homology with the one submission by scientist of Korea. 2 clones 98% homologous with Cs44. The one 60% homologous with SJCHGC06649 protein in Schistosoma japonicum. In addition, 7 clones homology with mitochondrion.We ligated the CS-gly gene to expression plasmid pET28a. After confirmation by PCR, restriction endonuclease digestion with EcoR I/Xho I and gene sequencing, the results of identification were all correct. The prokaryotic expression plasmids were transferred to E.coli (DE3) and induced by IPTG, analyzed existence and solubility of the CS-gly protein by SDS-PAGE. The SDS-PAGE show purpose bonds of CS-gly in expecting positions, whose relative molecular weights were 14.72kD. Western bloting confirmed the cs-gly protein was identified by rabbits-to-C.sinensis antiserum.This study lays basis for expression recombination of genes that encode high-reactionogenicity antigens and the set up immunological detection of Clonorchis sinensis which sensitive and specific.